10 Effect of blood

Biomédica 2006;26(Supl.1):57-63
EFFECT OF BLOOD SOURCE ON THE SANDFLY
ARTÍCULO ORIGINAL
Effect of blood source on the survival and fecundity of
the sandfly Lutzomyia ovallesi Ortiz (Diptera: Psychodidae),
vector of Leishmania
Pedro Noguera, Maritza Rondón, Elsa Nieves
Laboratorio de Parasitología Experimental (LAPEX), Departamento de Biología, Facultad de Ciencias,
Universidad de Los Andes, Mérida, Estado Mérida, Venezuela.
Introduction. The reproductive potential of sandflies depends on various factors, one of which
is the type of host available as blood source, which is important in determining their capacity to
serve as vectors.
Objective. The present study evaluated the effect of the animal blood source on various biological
parameters of Lutzomyia ovallesi (Ortiz) under laboratory conditions.
Materials and methods. Two-day-old females from a L. ovallesi colony were artificially fed to
repletion using a chicken skin membrane with blood from seven different species of vertebrate
hosts, horse, dog, cow, chicken, goat, pig and human. Life-span, time of oviposition, time for
blood digestion, number of eggs laid, number of eggs retained and the total number of eggs
were recorded.
Results. The results show the influence of blood source on different biological parameters of L.
ovallesi. The results showed that in L. ovallesi, chicken blood is the most quickly digested (3.34
days) and gives the longest time of oviposition (5.88 days), the greatest number of eggs retained
(10.20 eggs per female) and the greatest fecundity (30.80 eggs per female) compared with the
other sources of blood studied. The most satisfactory animal blood source was chicken followed,
in descending order, by goat, cow, pig, human, dog and horse.
Conclusions. The data showed that, in bio-ecological terms, the best blood source for L. ovallesi
was chicken and the least satisfactory one was horse. These results contribute to the
understanding of the factors that influence the rearing of the sand fly L. ovallesi under laboratory
conditions, and of how dietary factors for adult sand flies affect their biological potential and
could have important consequences on the transmission of Leishmania.
Key words: Blood, Psychodidae, biology, fertility, Leishmania.
Efecto del tipo de sangre en la supervivencia y fecundidad del flebotomino Lutzomyia
ovallesi Ortiz (Diptera: Psychodidae) vector de Leishmania
Introducción. El potencial reproductivo de los flebotominos depende de varios factores, uno
de los cuales es el tipo de hospedador disponible como fuente sanguinea, este es importante
en determinar su capacidad de servir como vectores.
Objetivo. Se estudia el efecto de la fuente de alimentación sanguínea sobre varios parámetros
biológicos de L. ovallesi en condiciones de laboratorio.
Materiales y métodos. Se utilizaron hembras de dos días de edad de L. ovallesi de colonia,
alimentadas artificialmente a repleción usando membrana de pollo, con sangre de siete
hospedadores vertebrados, caballo, perro, vaca, gallina, chivo, cochino y humano. Se determinó
el tiempo de vida, tiempo de oviposición, tiempo de digestión sanguínea, número de huevos
puestos, número de huevos retenidos y número de huevos totales.
Resultados. Los resultados muestran la influencia de la fuente sanguínea sobre diferentes
parámetros biológicos de L. ovallesi estudiados. Los resultados demuestran que con la sangre
de gallina se obtienen mayor tiempo de oviposición (5,88 días), digestión más rápida (3,34
días), mayor número de huevos retenidos (10,20 huevos por hembra) y mayor fecundidad
(30,80 huevos por hembra) en comparación con los otros tipos de sangre. La sangre más
57
NOGUERA P., RONDÓN M.,NIEVES E.
Biomédica 2006;26(Supl.1):57-63
satisfactoria fue la de gallina seguida, en orden descendente, por las de chivo, vaca, cochino,
humano, perro y caballo.
Conclusión. Los datos muestran que la sangre de gallina es la mejor fuente sanguínea en
términos bio-ecológicos para L. ovallesi, y la sangre de caballo la menos adecuada. Los
resultados contribuyen al entendimiento de los mecanismos que influyen en las condiciones
de cría en el laboratorio del flebotomino L. ovallesi y también de cómo ciertos factores de la
dieta en los adultos afectan el potencial biológico y que podrían tener importante consecuencias
en la transmisión de Leishmania.
Palabras clave: sangre, Psychodidae, biología, fertilidad, Leishmania.
Sandflies (Diptera: Psychodidae) are hematophagic
insects that need bloodmeal to complete their
reproductive cycle. Only the females feed on blood
which provides proteins indispensable for egg
production (1-3). The reproductive potential of
sandflies, as other hematophagic insects, depend
mainly to find a host that will provide blood capable
to mature a significant number of eggs besides a
suitable site for laying them (4-7). The feeding
habits of the vectors constitute an important
aspect of their bionomics, affecting directly the
Leishmania transmission (8-10). It also was
demonstrated that the rate of blood meal digestion
in Phlebotomus langeroni varied according to the
source of the vertebrate blood and Leishmania
species involved (11).
Chaniotis 1967(12) noticed that Lutzomyia vexator
laid more eggs after feeding on lizards than on
snakes. Ward 1977 (13) reported that L.
flaviscutellata laid more eggs after feeding on
rodents than on humans. Ready 1979 (14)
observed that for L. longipalpis the number of eggs
matured with the blood of seven mammal hosts
increased in direct proportion to the weight of blood
ingested and its source, some blood seems to be
more nutritive than others; similarly, Benito De
Martin et al. 1994 (15) and Hanafi et al. 1999 (16)
showed for Phlebotomus that fecundity also
dependeds on the blood source. Nutritional quality
of blood varies between host species and may
influence egg productivity, reduce development
Corresponding:
Elsa Nieves, Laboratorio de Parasitologia Experimental
(LAPEX), Departamento de Biología, Facultad de Ciencias
Universidad de Los Andes, La Hechicera, Mérida, Estado
Mérida, 5101. Venezuela.
Teléfono: (02 74) 240 1244; fax: (02 74) 240 1286.
[email protected]
Recibido: 26/05/05; aceptado: 13/10/05
58
rates, longevity and fecundity of the insects (17).
For understanding the role of blood meal sources
on sandfly biology, physiology and Leishmania
transmission more field observations and
laboratory studies comparing egg productivity of
sandflies fed on differents hosts (17-19) are
necessary.
L. ovallesi (Ortiz) is one of the main vectors of
Leishmania braziliensis in Venezuela (20). It is
frequently found in the vicinity of human habitation and it is considered an anthropophilic species (21,22). However, L. ovallesi feeds upon a
variety of vertebrate hosts, and could be considered as an opportunistic species (23,24). Thus,
the present work studies the effect of blood from
different domestic animals on the fecundity of L.
ovallesi in laboratory conditions.
Materials and methods
Sandflies
Females of L. ovallesi were obtained from a closed
laboratory colony established with specimens
collected during 2001 at 1,360 m above sea level
in the locality of El Arenal, Ejido, Mérida state,
Venezuela. The colony was maintained in the
Laboratorio de Parasitología Experimental, at the
Universidad de Los Andes, Mérida, Venezuela,
with techniques described by Killick-Kendrick et
al. in 1977 (25), in an incubator at 25±1°C with a
relative humidity of 80±10%.
Blood sources
Blood from healthy animals was collected in tubes
containg 0.2 ml of citrate per ml of blood. The
blood sources were: horse (Equus caballus),
chicken (Gallus domesticus), pig (Sus scrofa
domestica), cow (Bos taurus), goat (Capra hircus),
dog (Canis familiaris) and human (Homo sapiens
sapiens).
Biomédica 2006;26(Supl.1):57-63
Artificial feeding
Two-day-old females were artificially fed to repletion using chicken membrane, with water circulating at a temperature of 39°C. Groups of ≈100
sand flies were exposed to the feeder over each
carton during 4 hours. In each fedeer blood was
mixed every 30-45 minutes using a pipette, to prevent cell sedimentation. Blood-fed sandflies were
separated, counted and individually placed into
glass tubes, mantained in an incubator at 25±1°C
with relative humidity of 80±10%, with 12 hours
of light and 12 hours of darkness. As a dietary
supplement they were given a 50% fresh sucrose
solution, renewed daily.
EFFECT OF BLOOD SOURCE ON THE SANDFLY
gestion time for chicken blood and all the others,
and between digestion time of horse blood and
blood from chicken, pig, cow, and goat (p<0.0005).
Oviposition time. Figure 2 shows the oviposition
time by L. ovallesi females fed on the blood of
seven vertebrate hosts. The longest oviposition
time was obtained with chicken blood and the
shortest, with goat and dog blood. In ascending
order, the time of oviposition for L. ovallesi with
the different sources of blood was as follows:
dog<goat<horse<human<pig<cow<chicken. Statistically significant differences (p<0.05) were observed for oviposition time for L. ovallesi fed on
chicken, goat, and dog blood.
Fecundity, oviposition time and survival
Daily observations were made to determine the
time of blood digestion which was estimated
checking each one of the females under the light
microscope to determine the presence of blood
in the midgut; the oviposition time, which
represents the time from blood feeding to egg
laying; and survival represents the longevity of
the females. After oviposition and death of the
females, the eggs were counted, the females
removed and dissected in PBS solution using a
stereoscopic microscope and the number of
retained eggs counted for determination of the
fecundity (the number of eggs laid and retained).
All results represents the average per female of a
minimum of 60 females and a maximum of 139
females.
Figure 1. Digestion time of L. ovallesi feeding on blood of
seven vertebrate hosts. The numbers in parentheses are
the range and the number below each blood source is the n
value.
Statistical analysis
One way analysis of variance (ANOVA) was used,
with a Tukey test for different N, and finally a
principal component analysis, using Minitab
Statistical Software (version 10), the statistics
program (version 6.0) and SPSS (version 8 in
Spanish).
Results
Digestion time. The digestion time by L. ovallesi
females fed on the blood of seven vertebrate hosts
is presented in figure 1. The digestion time for L.
ovallesi in ascending order of duration is as follows: chicken<pig<cow<goat<dog<human<horse
(figure 1). Statistical analysis showed that there
was only a significant difference between the di-
Figure 2. Oviposition time of L. ovallesi feeding on blood of
seven vertebrate hosts. The numbers in parentheses are
the range and the number below each blood source is the n
value.
59
NOGUERA P., RONDÓN M.,NIEVES E.
Biomédica 2006;26(Supl.1):57-63
Number of eggs laid. Figure 3 shows the average
number of eggs laid by L. ovallesi females fed on
the blood of seven vertebrate hosts. The lowest
average occurred with horse blood and the highest
with goat. The number of eggs laid in decreasing
order was as follows: goat>chicken>pig>dog>
human>cow>horse. No significant differences
were found between the groups.
Number of eggs retained. Figure 4 shows the
average number of eggs retained by L. ovallesi
females fed on blood of the seven vertebrate
hosts. The lowest number of eggs retained
occurred with pig blood and the highest with
chicken. The number of eggs retained in
increasing order was as follows: pig<cow<human
<goat<horse<dog<chicken. Statistical analysis
showed only a significant difference in the average number of eggs retained by L. ovallesi fed on
blood of chicken and those fed on pig blood
(p<0.01).
Fecundity. Figure 5 shows fecundity values (the
number of eggs laid and retained) for L. ovallesi
females fed on the blood of different vertebrate
hosts. The fecundity values in decreasing order
were as follows: chicken>dog>goat>pig>human
>horse>cow. Statistical analysis shows significant
differences in the fecundity of L. ovallesi fed on
chicken blood compared with the values obtained
from cow, horse, human and pig blood. There were
also significant differences between the values
Figure 4. Number of eggs retained by L. ovallesi feeding on
blood of seven vertebrate hosts. The numbers in parentheses are the range and the number below each blood source
is the n value.
Figure 5. Fecundity of L. ovallesi feeding on blood of seven
vertebrate hosts. The numbers in parentheses are the range
and the number below each blood source is the n value.
obtained from cow and chicken blood, and between
dog and goat blood (p<0.05).
Figure 3. Number of eggs laid by L. ovallesi feeding on blood
of seven vertebrate hosts. The numbers in parentheses are
the range and the number below each blood source is the n
value.
60
Life span. The average values for life span of L.
ovallesi fed on blood of the seven vertebrate hosts
were analyzed (figure 6). The longest life span
occurred with cow blood and the lowest, with dog.
The average life span of L. ovallesi fed on different
types of blood in decreasing order was as follows:
cow>chicken>pig>human>goat>horse>dog. Statistical analysis showed that there was a significant difference in the average values obtained
from dog blood and the other sources, and between
the average values obtained from horse and
chicken blood and from cow and dog blood
(p<0.05).
Biomédica 2006;26(Supl.1):57-63
Figure 6. Life span of L. ovallesi feeding on blood of seven
vertebrate hosts. The numbers in parentheses are the range
and the number below each blood source is the n value.
Figure 7. Principal component analysis of six variables associated with L. ovallesi feeding with blood from seven
vertebrate hosts.
Principal component analysis. Figure 7 shows
the principal component analysis of 6 variables
studied in L. ovallesi fed on blood of the seven
vertebrate hosts. This analysis was made on the
basis of data obtained after the third day of feeding
on blood. The figure is made up from two components which account for 63.35% of variation.
Discussion
Fecundity and longevity in sandflies are important
factors in determining their capacity to serve as
vectors. The results of the present work show that
in L. ovallesi, chicken blood is the most quickly
digested and gives the longest time of oviposition,
the greatest number of eggs retained and the
greatest fecundity compared with the other
EFFECT OF BLOOD SOURCE ON THE SANDFLY
sources of blood studied. These results agree with
those of other researchers, who suggest that blood
source modifies some biological parameters in
sandflies (14-16). Females of L. ovallesi fed on
dog blood showed significant differences, with the
shortest time of oviposition and the shortest life
span. Those fed on cow blood showed significant
differences in terms of lower fecundity with those
fed on chicken, dog and goat blood. L. ovallesi
fed on horse blood showed significant differences
between those fed on chicken and cow blood in
terms of a shorter life span, between those fed on
pig and goat blood in terms of longer digestion
time, and with those fed on dog blood in terms of
a longer life span.
Ready (14) suggests that these differences in egg
production by L. longipalpis fed on hamster and
human blood could be due to differences in redcell content and not to L-isoleucine deficiency in
human red cells, since adding it did not increase
the number of mature eggs. The small specific
differences among the seven vertebrate blood
sources would explain the results obtained for L.
ovallesi. These agree with those of Ready (14),
who showed that for L. longipalpis the amount and
composition of blood were the principal factors
that influenced egg-production in laboratory-bred
females. He suggests that physiological factors,
such as sugar feeding, size, autogeny and copulation, are less important.
The biological potential of Lutzomyia depends on
various factors, one of which is the type of host
available as a blood source, where specific
differences in blood values are related with the
synthesis and rupture of the peritrophic matrix,
and the synthesis of digestive enzymes. The
enzymatic processes of sandfly guts were shown
to function differently, when triggered by different
types of meals (8,19,26). The protease levels in
the gut of P. langeroni vary in accordance with
the blood of the vertebrate host (11). However,
the blood meals from different species of vertebrate
have no deleterious effect on the development
of Leishmania braziliensis or Leishmania
amazonensis in the gut of Lutzomyia migonei and
parasite development was compatible with
digestion, independent of the bloodmeal source
(10).
61
NOGUERA P., RONDÓN M.,NIEVES E.
Although, a high number of sandfly species have
been successfully colonized during the last
decade, the factors limiting their productivity and
fecundity in the laboratory are unknown. Also, information on basic aspects of the biology of the
sandfly vectors is important to understand the
efficiency of transmission of Leishmania and vector competence (27-29). The results of the present
work show the influence of blood source on different biological parameters of L. ovallesi, with
chicken blood having the most significant effect
on several of the parameters studied. However,
no single blood source provided optimum results
for all the variables studied. On the basis of the
principal component analysis, and taking into account ideal or most adequate values for a specific
blood source, they would be those that gave, in
order of importance: (1) longest life span, (2)
shortest oviposition time, (3) shortest digestion
time, and (4) greatest number of eggs in total
(fecundity). In this analysis, for L. ovallesi fed on
seven types of blood, the best in bioecological
terms would be chicken, and the least satisfactory,
horse; the most satisfactory in descending order
being as follows: chicken, goat, cow, pig, human,
dog, and horse.
Nutritional quality of blood varies between host
species, avian blood should be less nutritious than
that of mammals (17). Chicken erythrocytes are
nucleate and have a DNA content 31 times higher
than the contents found in humans (8,17). They
also have a lower hemoglobin content than
mammalian red cells and half hematocrit value in
comparison to mammals, total plasma protein levels in chickens are considerably lower than in dogs
and pigs. In addition, catabolism of nucleic acids
from chicken erythrocytes would presumably involve greater bioenergetic cost due to increased
production and active transport of uric acid, the
end product of nitrogen metabolism (17). In a
comparative study of the fecundity and survival
rates of P. papatasi fed on blood from eight species of mammals (human, horse, cow, pig, dog,
rabbits, guinea-pigs and hamsters) appreciable
differences were not detected (30). We found that
L. ovallesi can be fed on a variety of source blood,
and the chicken blood appears to be more
competent. Extrapolating these results, it could
62
Biomédica 2006;26(Supl.1):57-63
be assumed that the effect would be similar in
natural conditions, but these aspects need further
study to determine the relative importance from
chicken and other hosts as bloodmeal sources.
Acknowledgments
We thank Carlos Araque for assistance in the production in laboratory colony, Luis Chávez for his
help and cooperation, and to professors Efrain
Entralgo, Guillermo Bianchi and Paolo Ramoni for
guidance in statistical analyses; to professor Leida
Quintero for help in providing blood samples and
Irlanda Márquez for proofreading this article.
Conflict of interests
The authors declare that they have no competing
interests
Financial support
This study was supported by grant of CDCHTULA (Cod: C-1406-06-03-B) and CONICIT (Cod.:
S1-2000000818).
References
1. Adler S. Leishmania. Adv Parasitol 1964;2:35-96.
2. Killick-Kendrick R. Biology of Leishmania in
phlebotomine sandflies. En: Lumsden WH, Evans DA,
editors. Biology of the Kinetoplastida. London: Academic
Press; 1979. p.395-460.
3. Young DG, Duncan MA. Guide to the identification
and geographic distribution of Lutzomyia sand flies in
Mexico, the West Indies, Central and South America
(Diptera: Psychodidae). Mem Ann Ent Inst 1994;54:881.
4. Van Handel E. Metabolism nutrients in the adult mosquito. Mosquito News 1984;44:573-9.
5. Briegel H. Fecundity, metabolism, and body size in
Anopheles (Diptera: Culicidae), vector of malaria. J Med
Entomol 1990;27:839-50.
6. Briegel H, Horler E. Multiple blood meal as reproductive strategy in Anopheles (Diptera: Culicidae). J Med
Entomol 1993;30:975-85.
7. Kassem HA, Hassan AN. Ovarian development and
blood-feeding activity in Phlebotomus bergeroti Parrot
(Diptera: Psychodidae) from Egypt. Ann Trop Med
Parasitol 2003;97:521-6.
8. Schlein Y, Warburg A, Schnur LF, Shlomai J. Vector compatibility of Phlebotomus papatasi dependent
on differentially induced digestion. Acta Trop
1983;40:65-70.
9. Volf P, Svobodova M, Dvorakova E. Bloodmeal digestion and Leishmania major infections in Phleboto-
Biomédica 2006;26(Supl.1):57-63
mus duboscqi: effect of carbohydrates inhibiting midgut lectin activity. Med Vet Entomol 2001;15:281-6.
10. Nieves E, Pimenta PFP. Influence of vertebrate blood
meals on the development of Leishmania (Viannia)
braziliensis and Leishmania (Leishmania) amazonensis
in the sand fly Lutzomyia migonei (Diptera: Psychodidae). Am J Trop Med Hyg 2002;67:640-7.
11. Daba S, Mansour NS, Youssef FG, Shanbaky NM,
Shehata MG, el Sawaf BM. Vector-host-parasite
interelationships in leishmaniasis. III. Impact of blood
meal from natural vertebrate host on survival and the
development of Leishmania infantum and L. major in
Phlebotomus langeroni (Diptera: Psychodidae). J Egypt
Soc Parasitol 1997;27:781-94.
12. Chaniotis BN. The biology of California Phlebotomus
(Diptera: Psychodidae) under laboratory conditions. J
Med Entomol 1967;4:221-33.
13. Ward RD. The colonization of Lutzomyia flaviscutellata
(Diptera: Psychodidae), a vector of Leishmania
mexicana amazonensis in Brazil. J Med Entomol
1977;14:469-76.
14. Ready PD. Factors affecting egg production of laboratory-bred Lutzomyia longipalpis (Diptera: Psychodidae).
J Med Entomol 1979;16:413-23.
15. Benito-De Martín MI, Gracia-Salinas MJ, MolinaMoreno R, Ferrer-Dufol M, Lucientes-Curdi J. Influence of the nature of the ingested blood on the
gonotrophic parameters of Phlebotomus perniciosus
under laboratory conditions. Parasite 1994;1:409-11.
EFFECT OF BLOOD SOURCE ON THE SANDFLY
Lutzomyia ovallesi (Ortiz) (Diptera: Psychodidae)
ocasionado por Leishmania (Leishmania) amazonensis.
Parasitol Latinoam 2004;59:115-22.
21. Feliciangeli MD. La fauna flebotómica (Diptera, Psychodidae) en Venezuela: 1. Taxonomía y distribución
geográfica. Bol Dir Malariol San Amb 1988;28:99-113.
22. Feliciangeli MD. Vector of leishmaniases in Venezuela. Parassitologia 1991;33:229-36.
23. Añez N, Cazorla D, Nieves E, Chataing B, Castro M,
De Yarbuh AL. Epidemiología de la leishmaniasis
tegumentaria en Mérida, Venezuela. I. Diversidad y
dispersión de especies flebotominas en tres pisos
altitudinales y su posible role en la transmisión de la
enfermedad. Mem Inst Oswaldo Cruz 1988;83:45563.
24. Noguera P, Chaves L, Nieves E. Effects of blood
ingestion on patterns on the chorion of eggs of
Lutzomyia ovallesi (diptera: psychodidae). Parasitol
Latinoam 2003;58:49-53.
25. Killick-Kendrick R, Leaney AJ, Ready PD. The establishment, maintenance and productivity of laboratory
colony of Lutzomyia longipalpis (Diptera: Psychodidae).
J Med Entomol 1977;13:429-40.
26. Schlein Y, Jacobson RL. Resistance of Phlebotomus
papatasi to infection with Leishmania donovani is
modulated by components on the infective bloodmeal.
Parasitol 1998;117:467-73.
16. Hanafi HA, Kanour WW Jr, Beavers GM, Tetreault
GE. Colonization and bionomics of the sandfly
Phlebotomus kazeruni from Sinai, Egypt. Med Vet
Entomol 1999;13:295-8.
27. Nieves E. Problemas de colonización de especies
flebotominas bajo condiciones de laboratorio, con especial referencia a Lutzomyia youngi , Lutzomyia
ovallesi y Lutzomyia migonei (trabajo para ascender
de categorí). Mérida, Venezuela: Universidad de los
Andes;1995. p.113.
17. Alexander B, Carvalho RL, McCallum H, Pereira MH.
Role of the domestic chicken ( Gallus gallus) in the
epidemiology of urban visceral leishmaniasis in Brazil.
Emerg Infect Dis 2002;8:1480-5.
28. Montoya J, Cadena H, Jaramillo C. Rearing and colonization of Lutzomyia evansi (Diptera: Psychodidae), a
vector of visceral leishmaniasis in Colombia. Mem Inst
Oswaldo Cruz 1998;93:263-8.
18. Agrela I, Sánchez E, Gómez B, Feliciangeli MD.
Multiple blood meals as a reproductive strategy in
Anopheles (Diptera:Culicidae). J Med Entomol 2001;
30:975-85.
29. Luitgards-Moura JF, Castellon EG, Rosa-Freitas
MG. Aspects related to productivity for four generations of a Lutzomyia longipalpis laboratory colony. Mem
Inst Oswaldo Cruz 2000;95:251-7.
19. Hurd H. Manipulation of medically important insect vectors by their parasites. Annu Rev Entomol 2003;48:14161.
30. Harre JG, Dorsey KM, Armstrong KL, Burge JR,
Kinnamon KE. Comparative fecundity and survival
rates of Phlebotomus papatasi sandflies membrane fed
on blood from eight mammal species. Met Vet Entomol
2001;15:189-96.
20. Nieves E, Dávila-Vera D, Palacios-Prü E. Daño
ultraestructural del intestino medio abdominal de
63